【摘要】 目的构建癫痫模型大鼠海马凋亡神经元的cDNA文库,采用酵母三杂交技术从中寻找caspase-3潜在的酶切底物。方法制作海人藻酸大鼠癫痫模型,取其海马组织制作酵母三杂交cDNA文库,克隆获得caspase-3的P12、P17两亚基,然后分别定向插入同一pBridge质粒,构建caspase-3的酵母三杂交诱饵载体并进行验证,然后对所构建文库进行筛库。结果成功构建癫痫模型大鼠海马组织cDNA文库,构建了caspase-3酵母三杂交诱饵载体,并通过caspase-3与eIF2α之间的相互作用验证,经筛库获得caspase-3的底物MIZ1。结论酵母三杂交技术可用于寻找caspase-3下游底物,从癫痫模型大鼠海马的cDNA文库中筛库获得caspase-3的底物MIZ1,为进一步研究caspase-3在癫痫发作引起的海马神经元损伤中的作用奠定了基础。更多还原

【Abstract】 Objective To construct the yeast three-hybrid cDNA library with hippocampi of epileptic model rats and to screen the library with the yeast three-hybrid system for latent substrates of caspase-3.Methods The yeast three-hybrid cDNA library was constructed with hippocampi of epileptic model rats.P12 and P17 subunits of caspase-3 were obtained by PCR and subcloned into the Multiclone Sites(MCS) of a pBridge for screening caspase-3 interacted substrates using yeast three-hybrid system.Feasibility of the bait vector was verified with eIF2 α and the library was screened with the bait vector for this novel substrates of caspase-3.Results Construction of the yeast three-hybrid cDNA library with hippocampal neurons of epileptic model rats was accomplished.The bait vector in yeast three-hybrid system for caspase-3 was constructed.And a novel substrate of caspase-3 was harvested.Conclusions The bait vector is feasible.With the bait we are able to verify that caspase-3 can interact with eIF2α.MIZ1 was cloned as a novel substrate of caspase-3 in the hippocampus of epileptic model rat,therefor it may play a role in hippocampal neuronal apoptosis after status epilepsy.更多还原